This invention pertains to four novel ubiquitin promoters derived from ubiquitin genes isolated from rice (Oryza sativa L.), promoters that efficiently drive constitutive gene expression in transgenic plants.
Significant advances in cell biology and gene delivery techniques have allowed incorporation of foreign genes into many crop plants. Foreign genes are transferred into plants, named xe2x80x9ctransgenic plants,xe2x80x9d primarily to express proteins that will confer a beneficial trait, such as resistance to pathogenic micro-organisms or insects, resistance to herbicides, or tolerance to drought or other adverse environrments. Usually, the DNA coding region for the foreign gene is linked to a strong and constitutive DNA promoter region to ensure the efficient expression of the foreign gene in the transgenic cell.
A number of common promoters are used to drive foreign gene expression in transgenic plants. The cauliflower mosaic virus (CaMV) 35S promoter has been widely used in both dicots and monocots, but its effectiveness in monocots was found to be substantially less than in dicots. CaMV 35S was even inactive in certain cell types, such as pollen. See Guerrero et al., xe2x80x9cPromoter sequences from a maize pollen-specific gene direct tissue-specific transcription in tobacco,xe2x80x9d Mol. Gen. Genet, vol. 224, pp. 161-168 (1990).
The maize alcohol dehydrogenase (Adh1) promoter has also been used in monocot transformation studies. This promoter has been shown to be 10 to 20 times more active than the CaMV 35S promoter in transformed rice protoplasts and cultured cells; however, the maize Adh1 promoter was not consitutively active in all transformed tissues. This promoter was induced by anaerobic stress in the transformed rice protoplasts. See Zhang et al., xe2x80x9cEfficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in the plants,xe2x80x9d Theor. App. Genet., vol. 76, pp. 835-840 (1988).
Promoters can be more effective if isolated from the same species as the transgenic plant. xcex2-glucuronidase (GUS) expression under the control of a rice actin promoter (Act1) in transformed rice protoplasts was approximately 6-fold greater than expression under control of the maize Adh1 promoter. Activity of the rice actin promoter is dependent on the presence of an intact Act1 5xe2x80x2 intron; i.e., removal of the intron resulted in no gene expression. See McElroy et al., xe2x80x9cIsolation of an Efficient Actin Promoter for Use in Rice Transformation,xe2x80x9d The Plant Cell, vol. 2, pp. 163-171 (1990).
Ubiquitin is one of the most highly conserved proteins in eukaryotes. See Callis et al., xe2x80x9cUbiquitin and Ubiquitin Genes in Higher Plants,xe2x80x9d Oxford Surveys of Plant Molecular and Cell Biology, vol. 6, pp. 1-30 (1989). One physiological role for ubiquitin is to conjugate with a target protein as a recognition signal for protein degradation. See Viersta, R.D., xe2x80x9cProteolysis in plants: mechanisms and functions,xe2x80x9d Plant Molecular Biology, vol. 32, pp. 275-302 (1996). In higher organisms, ubiquitin has been shown to be encoded by two small gene families, named xe2x80x9cpolyubiquitin genesxe2x80x9d and xe2x80x9cubiquitin fusion genes.xe2x80x9d Polyubiquitin genes comprise tandem head-to-tail repeats of 228 bp, with each repeat encoding 76 amino acids of a ubiquitin monomer. The number of tandem repeats reported varies between genes within genomes and between organisms, from 3 in Dictostylium to approximately 50 in Trypanosoma cruzi. On the other hand, the ubiquitin fusion gene family encodes a single repeat fused to one of two other polypeptides of either 52 or 76-80 amino acids. See Callis et al., xe2x80x9cUbiquitin and Ubiquitin Genes in Higher Plants,xe2x80x9d Oxford Surveys of Plant Molecular and Cell Biology, vol. 6, pp. 1-30 (1989). Studies of ubiquitin genes in a number of plants indicate that ubiquitin genes are expressed in all tissues; however, differential expression of the ubiquitin genes is also indicated among the ubiquitin gene family. Each tandem repeat or ubiquitin gene may be expressed differently over time and in different cells or tissues. Examples are given below.
The conditions that cause genetic expression of four ubiquitin-encoding cDNAs, including one ubiquitin fusion cDNA and three polyubiquitin cDNAs with 6 or 7 repeats, have been characterized in potato tuber. See Garbarino et al., xe2x80x9cExpression of stress-responsive ubiquitin genes in potato tubers,xe2x80x9d Plant Molecular Biology, vol. 20, pp. 235-244 (1992). The ubiquitin fusion cDNA encoded a single ubiquitin unit fused to an 80 amino acid ribosomal extension protein. Expression of the ubiquitin fusion gene was induced by injury or ethylene, but not by heat. Expression of the three polyubiquitin genes differed: one was induced by injury, heat, or ethylene treatment; another was induced by injury or heat, but not by ethylene treatment; and the remaining gene was expressed at the highest level, but its expression decreased in response to injury, heat, or ethylene treatment.
Expression of the ubiquitin gene families may be dependent on the type and age of the plant tissue, as well as certain environmental factors. A polyubiquitin gene from Nicotiana tabacum, Ubi.U4, was expressed throughout the plant, except in just-fully-expanded leaves. See Genschik et al., xe2x80x9cSturcture and promoter activity of a stress and developmentally regulated polyubiquitin-encoding gene of Nicotiana tabacum,xe2x80x9d Gene, vol. 148, pp. 195B-202 (1994). In tomato, expression of a ubiquitin fusion gene, ubi3, was highest in young leaves and immature green fruits and lowest in mature leaves and petals; however, expression was reduced by heat or light deprivation. See Hoffman et al., xe2x80x9cIsolation and characterization of tomato cDNA and genomic clones encoding the ubiquitin gene ubi3,xe2x80x9d Plant Molecular Biology, vol. 17, pp. 1189-1201 (1991). In parsley, expression of one polyubiquitin gene, ubi4, was predominant and was at comparable levels in all plant organs tested. See Kawalleck et al., xe2x80x9cPolyubiquitin gene expression and structural properties of the ubi4-2 gene in Petroselinum crispum,xe2x80x9d Plant Molecular Biology, vol. 21, pp. 673-684 (1993).
Promoters from ubiquitin genes have been shown to drive reporter gene expression, usually GUS or chloramphenicol acetyl transferase (CAT), in transformed cells or plants. Such promoters have been isolated from Arabidopsis (Callis et al., xe2x80x9cUbiquitin Extension Proteins of Arabidopsis thaliana,xe2x80x9d The Journal of Biological Chemistry, vol. 265, no. 21, pp. 12486-12493 (1990)); sunflower (Binet et al., xe2x80x9cAnalysis of a sunflower polyubiquitin promoter by transient expression,xe2x80x9d Plant Science, vol. 79, pp. 87-94 (1991)); tobacco (Genschick et al., xe2x80x9cStructure and promoter activity of a stress and developmentally regulated polyubiquitin-encoding gene of Nicotiana tabacum,xe2x80x9d Gene, vol. 148, pp. 195-202 (1994)); and maize (Christensen et al., xe2x80x9cMaize polyubiguitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation,xe2x80x9d Plant Molecular Biology, vol. 18, pp. 675-689 (1992)).
The ubiquitin promoter ubil isolated from a maize polyubiquitin gene was shown to drive the expression of the CAT reporter gene more efficiently than the CaMV 35S promoter in maize protoplasts. See Christensen et al., xe2x80x9cMaize polyubiguitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation,xe2x80x9d Plant Molecular Biology, vol. 18, pp. 675-689 (1992). The maize ubil-promoter has been used to express a herbicide resistance gene in rice. See Toki et al., xe2x80x9cExpression of a Maize Ubiquitin Gene Promoter-bar Chimeric Gene in Transgenic Rice Plants,xe2x80x9d Plant Physiol, vol. 100, pp. 1503-1507 (1992).
U.S. Pat. Nos. 5,614,399 and 5,510,474 describe a promoter from a maize polyubiquitin gene. The promoter regulates expression of a maize polyubiquitin gene containing 7 tandem repeats. Expression of this maize ubiquitin gene was constitutive at 25xc2x0 C., and was induced by heat shock at 42xc2x0 C. The promoter was successfully tranformed and expressed in other monocot plants in addition to maize, including wheat, barley, and rice. See Christensen et al., xe2x80x9cUbiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants,xe2x80x9d Transgenic Research, vol. 5, pp. 213-218 (1996). In contrast, expression of this maize promoter in idicot plants was relative low. For example, the CAT reporter gene expression under control of a maize ubiquitin promoter was 10 times less than that under control of the CaMV 35S in tobacco protoplasts. See Christensen et al., Plant Mol. Biol., vol. 18, pp. 675-689 (1992). In the maize ubil promoter region, a TATA box was found at position of xe2x88x9230, and two overlapping heat shock sequences, 5xe2x80x2-CTGGTCCCCTCCGA-3xe2x80x2 (SEQ ID NO 13) and CTCGAGATTCCGCT-3xe2x80x2(SEQ ID NO 14), were found at positions xe2x88x92214 and xe2x88x92204. The canonical CCAAT and the GC boxes were not found in the promoter region, but the sequence 5-CACGGCA-3xe2x80x2 (function unknown) occurred four times, at positions xe2x88x92236, xe2x88x92122, xe2x88x9296, and xe2x88x9291 of the promoter region. See Christensen et al., xe2x80x9cMaize polyubiguitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation,xe2x80x9d Plant Molecular Biology, vol. 18, pp. 675-689 (1992).
U.S. Pat. No. 5,773,705 describes a method to use ubiquitin monomers to enhance gene expression. The polyubiquitin gene was translated as the precursor polyubiquitin protein. The precursor polyubiquitin protein was then split into a 76 amino-acid ubiquitin monomer. If a single repeat of the ubiquitin DNA sequence was added upstream of a structural gene, a fusion protein was produced. The ubiquitin monomer was then cleaved from the fusion protein to release the protein encoded by structural genes. This method to enhance gene expression was documented for several polyubiquitin genes.
U.S. Pat. No. 5,723,757 describes a method to increase a tissue-specific (the storage or sink organ) expression of a desired DNA sequence by linking the DNA sequence to either a class I patatin promoter or a B33 promoter.
U.S. Pat. No. 5,723,765 describes a method of creating a transgenic plant that contains a gene whose expression can be controlled by application of an external stimulus. U.S. Pat. No. 5,750,866 describes a maize AHAS promoter used for heterologous gene expression in plants.
Rice is one of most important crops in the world, and is a model plant for genetic engineering. Strong promoters isolated from the rice genome will facilitate genetic improvement of rice. Additionally, such promoters may be effective in other plants, monocots and dicots.
We have sequenced four rice ubiquitin genes, including the promoter region for each. Of the four genes, two belong to the family of polyubiquitin genes, designated RUBQ1 and RUBQ2; and the other two belong to the ubiquitin fusion gene family, designated RUBQ3 and RUBQ4. The two polyubiquitin genes both comprise 6 ubiquitin-monomers in the coding region. Expression of the polyubiquitin gene, RUBQ2, was high in all rice plant tissues that were tested. Expression of the RUBQ2 gene was also induced by heat-shock treatment. Promoters isolated from RUBQ1 and RUBQ2 genes were shown to drive strong and constitutive expression of foreign genes in transformed rice plants.
The two promoters from the polyubiquitin genes were much more effective than the maize ubiquitin promoter in gene expression in rice. GUS expression under control of either RUBQ1 or RUBQ2 promoter was much higher than that under control of the CaMV 35S or maize ubiquitin promoter. Additionally, transgenic rice plants stably expressing the GUS gene under control of the RUBQ1 or RUBQ2 promoter have been developed. Strong GUS expression in roots was observed. However, GUS expression in roots of transgenic plants derived from transformation with CaMV 35S promoter-GUS construct was not readily detectable.